PROJECT SUMMARY CADA compounds selectively decrease expression of cell-surface CD4 on human immune cells. They are of interest as HIV entry inhibitors and for chemotherapy of various immune system-based diseases involving CD4+ T-cells, including asthma, rheumatoid arthritis, and diabetes. CADA down-modulates cell- surface CD4 by a novel mechanism of action, directly binding the signal peptide of nascent CD4 and inhibiting its translocation across the membrane of the endoplasmic reticulum (ER). It is the only small molecule known to specifically inhibit co-translational translocation of any protein across the ER membrane by binding its signal peptide. Hence, CD4 down-modulators may unlock a novel approach to suppressing other cell-surface proteins of therapeutic interest. The overall goals of the proposed project are: 1) to determine the molecular details of how CADA compounds bind the CD4 signal peptide and prevent passage of the CD4 protein through the channel across the ER membrane during translation; and, 2) to identify compounds that may be used to test the effect of CD4 down-modulation in animal models of the human immune system. In this project, we will synthesize PEGylated CADA analogs to improve their water solubility and bioavailability in order to produce better drug candidates and compounds that can be used to measure affinity for the CD4 signal peptide in aqueous media. We will also synthesize CADA analogs bearing photoreactive groups in order to characterize the binding site of the CD4 signal peptide by photoaffinity labeling. Photoaffinity labeling experiments will be conducted with the CD4 signal peptide in in solution, immobilized on a surface, and inserted in the ER transmembrane channel. Amino acid residues in the binding site of the CD4 signal peptide will be identified by mass spectrometry. These results will be used to develop a computational model of the complex between CADA compounds and the CD4 signal peptide. We will use this model to design target CD4 down-modulators based on novel molecular scaffolds, including pyridine-fused CADA analogs and cyclic tripeptides and tetrapeptides. The computationally screened compounds will be synthesized by conventional methods and also by new synthetic approaches to functionalized pyridines and small cyclic peptides. These new approaches would enable the synthesis of many new drug candidates for diverse human diseases. The new compounds will be tested for CD4 down-modulation potency by collaboration with the Rega Institute for Medical Research at the University of Leuven, Belgium. Potent compounds will undergo initial in vitro pharmacokinetic evaluation at UNR for water solubility, lipophilicity, cell permeability, and metabolic stability. The compounds with the best properties (ca. 5) will then undergo in vivo measurements of toxicity, oral bioavailability, and blood plasma lifetime in mice at the Rega Institute. Development of CD4 down-modulators that can be studied in vivo will enable future evaluation of drug efficacy and safety, and will make it possible to perform research aimed at revealing the detailed roles of the CD4 molecule in the human immune system.